This application focuses on African trypanosomes of the Trypanosoma brucei subspecies, the causative agents of human African trypanosomiasis, a fatal disease unless treated, with a calculated disease burden of about two million Disability Adjusted Life Years. There are no vaccines and the few available therapeutic drugs have serious side effects and decreasing efficacy in light of the emergence of drug-resistant trypanosomes. Our interest in preventing and curing parasite infections is focused on understanding and ultimately exploiting genetic mechanisms that are essential for all stages of the parasite life cycle, but are either absent or fundamentally different in the human host. One possible therapeutic approach is suggested by the observation that trypanosomatids have strikingly unusual mRNA biosynthetic pathways. A key molecule in this process is the RNA polymerase (pol) II-transcribed spliced leader (SL) RNA, because its m7G-capped 5' terminal sequence is trans-spliced onto the 5' end of each mRNA. Furthermore, a subset of small nuclear RNAs involved in RNA processing, including U1, U2, U3 and U4, are also capped, but they are unusual in that the corresponding genes are transcribed by pol III. In the last funding period our work has led to the identification of two pathways that are essential for mRNA metabolism and viability in T. brucei. First, m7G capping of the SL RNA is carried out by a dedicated capping enzyme, TbCGM1, whose depletion leads to cell death. Second, the hypermodified cap 4 structure of mature SL RNA is specifically recognized by a nuclear cap-binding complex, which is unique in the eukaryotic kingdom and whose depletion is also incompatible with life. Furthermore, we have made the first step towards defining the cellular function of the previously identified capping enzyme TbCE1, namely m7G capping of some of the U-snRNAs; this is a distinctive trait not found in any other eukaryotic organism. Most interestingly, the N-terminal domain of TbCE1 harbors a metal-independent RNA triphosphatase activity with no structural or functional similarities to the previously characterized T. brucei RNA triphosphatase TbCet1 and both triphosphatases are fundamentally different from the triphosphatase present in the human host. The long- term goal of this proposal is to understand the parasite-specific biology and mechanisms of RNA capping in T. brucei and to identify essential factors, factor domains, or protein-protein interactions which might be exploited for parasite control. In the next funding period we plan to: 1. Carry out a functional analysis of the SL RNA m7G capping machinery and the novel metal-independent triphosphatase domain of TbCE1. 2. Identify determinants that enable selective capping of the pol II-transcribed SL RNA and a subset of pol III transcripts. 3. Further characterize the nuclear cap binding complex to elucidate the basis for its specificity for the cap 4 structure and define its in vivo function more precisely. PUBLIC HEALTH RELEVANCE: Parasitic protozoa are a major cause of global infectious diseases and thus, represent one of the most serious threats to public health. Among these are African trypanosomes, the causative agents of African trypanosomiasis or sleeping sickness in humans and a wasting and fatal disease (Nagana) in cattle, domestic pigs and other farm animals causing a profound effect on the economy of much of the continent. Unless treated, African sleeping sickness is always fatal; no vaccine has been approved and there is a very limited arsenal of drugs with generally severe shortcomings, such as high toxicity and emerging resistance.